The Role of Angiotensin Converting Enzyme (ACE)

189 Pages·2016·2.47 MB·English

by  Nathan Michael Weir

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Nathan Michael Weir
Wright State University
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The Role of Angiotensin Converting Enzyme (ACE) 2 in a Murine Model of Insulin
Resistance and Albuminuria

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor
of Philosophy

By

Nathan Michael Weir
B.S. The Ohio State University, 2005

2012
Wright State University
COPYRIGHT BY
NATHAN MICHAEL WEIR
2012
WRIGHT STATE UNIVERSITY
SCHOOL OF GRADUATE STUDIES

May 11, 2012
I HEREBY RECOMMEND THAT THE DISSERTATION PREPARED UNDER MY
SUPERVISION BY Nathan Michael Weir ENTITELD The Role of Angiotensin Converting
Enzyme (ACE) 2 in a Murine Model of Insulin Resistance and Albuminuria BE ACCEPTED IN
PARTIAL FULFILLMENT OF THE REQUIREMETNS FOR THE DEGREE OF Doctor of
Philosophy
--------------------------------------------
Mariana Morris, Ph.D.
Dissertation Co-Director

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Khalid Elased, Pharm. D., Ph.D.
Dissertation Co-Director

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Gerald Alter, Ph.D.
Director, Biomedical Sciences Ph.D. Program

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Andrew Hsu, Ph.D.
Dean, School of Graduate Studies
Committee on Final Examination

------------------------------------------
Mariana Morris, Ph.D.

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Khalid Elased, Pharm. D., Ph.D.

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Paul Koles, M.D.

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Mark Anstadt, M.D.

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Christopher Wyatt, Ph.D
ABSTRACT OF DISSERTATION
Nathan Michael Weir. Biomedical Sciences Ph.D. Program, Wright State University, 2012; Co-
Advisors: Mariana Morris, Ph.D., Khalid Elased, Pharm. D., Ph.D., FAHA

The Role of Angiotensin Converting Enzyme (ACE) 2 in a Murine Model of Insulin
Resistance and Albuminuria.

Insulin resistance and kidney disease are intimately linked and both independently tied to
dysregulation of the renin angiotensin system (RAS). The purpose of this research was to better
investigate Angiotensin Converting Enzyme (ACE) 2 as a possible motif in the pathophysiology
of these two disease processes. The project was divided into two parts. Part 1 investigated ACE2
KO mice as a model of albuminuria. Part 2 investigated how the lack of ACE2 enzyme affected
glucose homeostasis and subsequent albuminuria.

Part 1: The Role of ACE2 in the Progression of Albuminuria
To determine if global loss of ACE2 leads to spontaneous development of albuminuria in mice,
we followed male wild type (WT) and knockout (KO) mice until 20 weeks of age. Basic
metabolic parameters and blood pressure were measured in addition to urinary parameters, and
histological evaluation. We found ACE2 KO mice only differed from their ACE2 WT mice
littermates in terms of increased water consumption which correlated with increased urine output.
Systolic blood pressure was also found to be mildly increased during the dark period. Urinary
albumin was similar between WT and KO mice as was total protein and sodium excretion.
Despite the lack of ACE2 in KO mice, urinary angiotensin (Ang) II and Ang-(1-7) were found to
be equal to that of ACE2 WT mice. Reduction-oxidation (redox) studies suggested elevated
oxidative stress in ACE2 KO kidneys though histopathology did not reveal any major changes at
the tissue level.
To determine the role of ACE2 in a model of kidney disease, mice were chronically infused with
Ang II. Both WT and KO mice groups had a similar pressor response, but ACE2 deficient mice
showed higher levels of kidney damage with respect to albuminuria and proteinuria. In
concordance, integrity of the glomerular filtration barrier was also diminished. Urinary Ang II
increased significantly in KO mice while Ang-(1-7) levels were raised equally. Renal pathology
revealed greater mesangial expansion and renal fibrosis in following Ang II infusion as well as
elevated redox status vs. the Ang II infused WT mice.
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To determine the role of the major ACE2 product Ang-(1-7), mice deficient in ACE2 were
infused with Mas receptor inhibitor, A779. The Mas receptor blockade did not result in
significant phenotypic or urinary differences compared to control groups. Urinary Ang levels
were also unchanged but ACE2 activity levels increased. This was linked to increased NADPH
oxidase (Nox) activity underscoring the role of the Mas receptor in Nox regulation. Overall, a
paucity of histological change was also observed despite increased Nox activity.
Together, these data suggest a lack of ACE2 does not precipitate albuminuria under normal
conditions. The similar urinary Ang levels point towards alternative pathways to Ang II
degradation and Ang-(1-7) synthesis which may influence Nox activity. Finally, ACE2 may
work as a compensatory enzyme to protect the kidney during times of Ang II excess.

Part 2: The Role of ACE2 in Glucose Homeostasis
To determine the role of ACE2 in obesity-related insulin resistance, ACE2 null mice were fed a
high fat (HF) diet for 12 weeks. Body weight and fat were continuously monitored and ACE2
KO mice gained weight at the same rate as WT mice littermates. Terminal fasting glucose and
glucose tolerance were found to be impaired in ACE2 KO mice despite similar weights and body
fat. Plasma metabolic hormones and adipokines were compared between groups. ACE2 KO
mice showed decreased plasma insulin concentration as well as higher cholesterol levels.
Pancreas ACE2 activity was elevated in control mice on a HF diet, suggesting a protective role of
ACE2 in the endocrine pancreas.
Next we investigated obesity-associated insulin resistance of ACE2 null mice as a model for
diabetic albuminuria. The level of insulin resistance and circulating blood glucose was not
sufficient to produce albuminuria in ACE2 KO mice after 12 weeks of HF diet. Renal histology
lacked mesangial expansion or decreased nephrin in the slit diaphragm. Kidney Nox activity was
found to be increased along with fibrosis. ACE2 activity in the kidney was increased among WT
mice as a result of HF diet.
Our results showed the ACE2 KO mouse model demonstrated impaired glucose tolerance likely
due to altered insulin secretion in the pancreas. However, the level and duration of glycemia
were not sufficient to produce a model of diabetic nephropathy.

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Table of Contents Page
Part 1. The Role of ACE2 in the Progression of Albuminuria 1
Section I: Introduction 1
1.1 Diabetes 1
1.1a Types of diabetes 1
1.2 Diagnosis 2
1.3 Diabetic complications 2
1.4 The Renin Angiotensin System 4
1.5 The Angiotensin II Receptors 6
1.5a AT R and hypertension 7
1
1.5b AT R and diabetes 8
1
1.6 Angiotensin Converting Enzyme 2 (ACE2) 9
1.7 The Mas Receptor 10
1.8 Adipose RAS 11
1.8a Renal RAS 12
1.9 ACE2 and renal dysfunction 13
1.10 Redox Status 15
1.11 AT and Reactive oxygen species (ROS) 17
1
1.12 Nephropathy 18
1.12a Diabetic nephropathy 19
1.13 Albuminuria Treatment Strategies 20
1.14 Glucose Control Strategies 21
Section II: Statement of the problem 25
Section III: Central hypothesis 27
3.1 Global ACE2 KO mice are protected against albuminuria 27
Section IV: Specific aims 28
4.1 Complete lack of ACE2 in a mouse model precipitates albuminuria 28
4.2 Infusion of Ang II exacerbates albuminuria in ACE2 null mice 28
4.3 Mas receptor antagonism exacerbates albuminuria in ACE2 KO mice 28
Section V: Materials and methods 29
5.1 ACE2 Mouse Genotyping 29
5.2 Animal housing and care 30
5.3 Osmotic pump surgery and infusion 30
5.4 Radiotelemetry surgery 30
5.5 Measurement of blood pressure 31
5.6 Metabolic urine collection 31
5.7 Urinary measurements 31
5.8 Blood and tissue collection 32
5.8a Perfusion 33
5.9 NAD(P)H oxidase activity 33
5.10 Picrosirius Red staining for collagen 34
5.11 Dihydroethidium (DHE) staining for ROS 34
5.12 Fluorescent microscopy 35
5.13 Western blot 35
5.14 Renal histology 35
5.15 Ang RIAs 36
5.16 Data and statistical analysis 37
Section VI: Results of specific aim 1 38
6.1 Basic metabolic and cardiovascular parameters 38
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6.1a Food, water, and urine 38
6.1b Baseline cardiovascular parameters 38
6.2 Kidney function 39
6.2a Creatinine 39
6.2b Albumin 40
6.2c Protein 40
6.2d Sodium 40
6.2e Ang peptides 40
6.3 Renal ACE2 expression and activity 41
6.4 Kidney histology 41
6.4a Morphology 41
6.4b Fibrosis 42
6.5 Nephrin immunofluorescence 41
6.6 Renal redox status 41
6.6a Superoxide anion 42
6.6b NADPH oxidase activity 42
Section VII: Results of specific aim 2 43
7.1 Basic metabolic and cardiovascular parameters 43
7.1a Food, water, and urine 43
7.1b Cardiovascular parameters 43
7.2. Kidney function 44
7.2a Creatinine 44
7.2b Albumin 44
7.2c Protein 44
7.2d Sodium 45
7.2e Ang peptides 45
7.3 Renal ACE2 activity 45
7.4a Morphology 45
7.4b Fibrosis 46
7.5 Nephrin immunofluorescence 46
7.6 Renal redox status 46
7.6a Superoxide anion 46
7.6b NADPH oxidase activity 46
Section VIII: Results of specific aim 3 47
8.1 Basic metabolic parameters 47
8.2 Kidney function 47
8.2a Creatinine 47
8.2b Albumin 47
8.2c Protein 48
8.2d Sodium 48
8.2e Ang peptides 48
8.3 Renal ACE2 activity 48
8.4 Kidney histology 48
8.4a Morphology 48
8.4b Fibrosis 48
8.5 Nephrin Immunofluorescence 49
8.6 Renal redox status 49
8.6a Superoxide anion 49
8.6b NADPH oxidase activity 49
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Section IX: Discussion 50
9.1 Overview 50
9.2 Morphometrics 50
9.3 Blood pressure 52
9.4 Urinary parameters 54
9.5 ACE2 in the kidney 57
9.6 Reactive oxygen species 58
9.7 Histology 60
Part 2: The Role of ACE2 in Glucose Homeostasis 62
Section I: Introduction 62
1.1 Obesity 62
1.2 Obesity and type 2 diabetes mellitus 62
1.3 ACE2 in adipose 64
1.4 ACE2 in the pancreas 65
Section II: Statement of the problem 66
Section III: Central hypothesis 67
Section IV: Specific aims 68
4.1 Specific aim 1 68
4.2 Specific aim 2 68
Section V: Materials and methods 69
5.1 ACE2 Mouse Genotyping 69
5.2 Animal housing and care 69
5.3 Metabolic urine collection 9
5.4 Urinary measurements 69
5.5 Glucose tolerance test 69
5.6 Blood and tissue collection 69
5.7 Plasma measurements 70
5.8 Morphometric analysis 70
5.9 ACE2 enzyme activity 70
5.10 NADPH oxidase activity 70
5.11 Fluorescent microscopy 71
5.12 Renal histology 71
5.13 Data and statistical analysis 71
Section VI: Results of specific aim 1 72
6.1 Basic metabolic 72
6.1a Caloric intake 72
6.1b Water consumption 72
6.1c Urine output 72
6.2 Body weight and percent body fat 72
6.3 Glucose homeostasis 73
6.3a Fed, fasted and glucose tolerance 73
6.4 Plasma hormones; insulin, glucagon 74
6.5 Plasma adipokines; adiponectin and leptin 75
6.6 Plasma lipids; triglycerides and cholesterol 75
6.7 Pancreas; ACE2 activity 76
Section VII: Results of specific aim 2 77
7.1 Urinalysis 77
7.1a Water intake and urine volume 77
7.1b Creatinine 77
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7.1c Albumin 77
7.1d Sodium 78
7.2 Histology 78
7.2a Morphology 78
7.2b Nephrin 78
7.3 Fibrosis 78
7.4 Renal ACE2 78
7.5 Redox status 78
7.5a Superoxide anion 78
7.5b Nox activity 79
Section VIII: Discussion 80
8.1 Caloric intake and body composition 80
8.2 Glucose homeostasis 82
8.3 Plasma hormones 83
8.4 Pancreas RAS 85
8.5 Urinary parameters 87
Perspective 90
Common abbreviations 129
References 131

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